Flagella promote swimming motility that is required by many pathogenic bacteria to navigate environments, infect hosts to promote disease, and promote biofilm formation. Construction of a flagellum requires the correct ordered expression of over 40 flagellar genes so that flagellar proteins are secreted and interact properly to facilitate organelle biosynthesis. As such, bacteria link steps in flagellar biogenesis to transcriptional regulation of distinct sets of flagellar genes. Historically, peritrichous organisms such as E. col and Salmonella species have served as models for understanding regulatory mechanisms for flagellar gene expression and biosynthesis. However, polarly-flagellated bacterial pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, Campylobacter jejuni, and Helicobacter pylori employ a different set of factors, including s54 and a flagellar-associated two-component regulatory system (TCS), to regulate expression of specific flagellar genes. Thus, new mechanistic paradigms must be developed to account for how polarly-flagellated bacteria regulate flagellar gene expression for flagellar biosynthesis. Through genetic and biochemical approaches, we discovered in Campylobacter jejuni and V. cholerae that flagellar substructures, including the flagellar type III secretion system, the MS ring and rotor component of the C ring, form a regulatory checkpoint during flagellar biosynthesis that is detected by the histidine kinases of the flagellar-associated TCSs. Subsequent signal transduction through the TCSs activates s54 and flagellar gene expression. This signaling pathway ensures the coordinated expression of flagellar genes that are conducive to form a flagellum to promote motility and flagellar-dependent activities required for infection of host, virulence, and biofilm formation. However, our analysis of C. jejuni and V. cholerae revealed that how flagellar structures form the regulatory checkpoint or how the checkpoint is detected by the TCSs show significant mechanistic variations. In Aim 1, we will perform genetic and biochemical analyses to assess the level of both potential general conservation and mechanistic variations of this signal transduction pathway for monitoring similar regulatory checkpoints during flagellar biogenesis in P. aeruginosa and H. pylori relative to C. jejuni and V. cholerae. In Aim 2, we will thoroughly analyze the signal transduction pathway in V. cholerae to understand how specific factors required in this bacterium, but absent in C. jejuni, have altered the mechanism by which the TCS monitors formation of the regulatory checkpoint formed by flagellar structures for proper expression of s54-dependent flagellar genes. Accomplishment of these aims will: 1) promote new understandings in signal transduction mechanisms required for flagellar gene expression in polarly-flagellated pathogens; 2) generate new knowledge regarding steps essential for flagellar biogenesis in motile bacteria that contribute to flagellar-dependent activities required for host infection of hosts; and 3) provide insights into mechanisms for how bacterial TCSs detect and perceive stimuli required to initiate signal transduction.